Cirsilineol improves anesthesia/surgery-induced postoperative cognitive dysfunction through attenuating oxidative stress and modulating microglia M1/M2 polarization

Ethics

All the animal experiments in this study were reviewed and approved by The Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine Animal Experimental Ethics Committee (SH9H-2024-A1018-SB) and performed according to the guidelines of China Council on Animal Care and Protocol and the ARRIVE guidelines. Effort has been made to minimize the suffering to the animals.

POCD mouse model construction

Aged male C57BL/6J mice (18 months old, body weight: 25–30 g) were purchased from the Shanghai Experimental Animal Research Center (Shanghai, China) and reared in groups (22–25 °C) under a 12-h circadian cycle and provided with free access to water and food.

All the mice were randomly grouped into Sham, Anesthesia (A)+Surgery (S)+ phosphate buffered saline (PBS) (A+S+PBS) (anesthesia and surgical management, and PBS was given), and A+S+CSL (anesthesia and surgical management, and received cirsilineol treatment) (n = 6 for each group) and unbiased double-blinded experiments were performed during the daytime. Specifically, the mice in Sham group only received the anesthesia and analgesia without any other surgery procedure; the mice in the A+S+PBS group were given equal volume of PBS solution before the anesthesia and surgery; the mice in the A+S+CSL group were given cirsilineol via intraperitoneal injection at 10 mg/kg for seven continuous days before the anesthesia and surgery. In this study, 10% dimethyl sulfoxide (DMSO, ST038; Beyotime, Shanghai, China), 40% PEG300 (HY-Y0873; MedChemExpress, Monmouth Junction, NJ) and 50% saline were used to dissolve Cirsilineol (HY-119347; MedChemExpress, Monmouth Junction, NJ, USA) as needed.

The POCD mouse model was established by referring to a previous study (Liu et al., 2021). Following 1-week acclimation, the mice were given 3% isoflurane (C153359; Aladdin, Shanghai, China) for anesthesia induction and 1.5% isoflurane for maintenance. An incision was made on the lateral side of the left tibia to expose the bone. An intramedullary fixation pin (0.3 mm) was implanted to the spinal canal from the hole drilled at the trochanter of the tibia. Next, osteotomy was performed at the middle and distal tibia, and the incision was closed with 5-0 Vicryl sutures. The body temperature of mice was monitored and maintained at 37 ± 0.5 °C using a heating pad during the operation. The mice were put back to the home cages after they had recovered from anesthesia and surgery. A total of 2% lidocaine (Shiyao Yinhu Pharmaceutical Co., Ltd., Sichuan Sheng, China) was locally applied for the treatment of postoperative pain twice per day for three successive days.

Behavioral tests

The behavioral tests in the current study included Morris water maze test and Y-maze test. For the Morris water maze test, a circular pool (depth: 50 cm, diameter: 120 cm) pre-filled with water (depth: 38 cm) was painted with tempera for spatial cues. A platform (diameter: 10 cm) was placed 1 cm below the water (water temperature was set at room temperature of 22 °C). In the training phase, the mice were instructed to familiarize themselves with water and swimming for 3 days (five training sessions per day) and had to locate the platform within 1 min. Then the mice were allowed to rest on the platform for 15 s. Those who failed to find the platform within 1 min were guided to the platform and also allowed to rest on the platform for 15 s. For the testing phase, the platform was removed and the mice were allowed to swim freely in the pool for 90 s in a total of 4 days. The relevant trajectory was recorded and the escape latency (s) was calculated by the experimenters blinded to the allocations of the animals (Wei et al., 2021).

For the Y-maze test, a Y-maze apparatus (40 cm × 5 cm × 10 cm) with two equal arms and one different arm separated by 120° was applied (Kraeuter, Guest & Sarnyai, 2019). In the first part of the test, one of the arms were blocked from the opening ones with an opaque board with the same texture of the apparatus. The mice were put at the end of the arm and allowed to explore the unblocked arms for 8 min. After 1 h, the blocking board was removed to allow the mice to explore for another 8 min as the second part of the test. The spontaneous alternation (SA) rate (%) was calculated by the experimenters blinded to the allocations of animals using the following formula: SA (%) = [number of alternations/(total number of arm entries -2)] × 100%.

At the end of the experiments, all the mice were sacrificed via inhalation of CO₂ overdose, and the samples were collected for subsequent use.

Cell culture and intervention

Dulbecco’s modified Eagle’s medium (DMEM, 11966-025; Gibco, Grand Island, NY, USA) added with 10% fetal calf serum (10099-141C; Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin (15070-063; Gibco, Waltham, MA, USA) was used to culture the murine microglia cell line BV-2 (CL-0493; Procell, Wuhan, China) at 37 °C with 5% CO₂. Next, the cells were grouped as follows: (1) Control (Con): BV-2 cells were normally cultured without intervention; (2) H₂O₂: BV-2 cells were exposed to 100 μM H₂O₂ for 24 h; (3) H₂O₂ + CSL: BV-2 cells were pre-treated with cirsilineol (5, 10, 20, 40 and 80 μM) for 24 h and exposed to 100 μM H₂O₂ for another 24 h.

Cell viability test

The Cell Counting Kit-8 (CCK-8) assay kit (C0037; Beyotime, Beijing, China) was applied for the cell viability test. BV-2 cells of each group were seeded into 96-well plates at the density of 5 × 10³ cells per well and supplemented with 10 μL CCK-8 solution for 3-h culture at 37 °C. A microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used for reading at 450 nm.

Measuring the ROS content

The cellular ROS content in BV-2 cells of each group was quantified with a commercial assay kit (E-BC-K138-F; Elabscience, Hoston, TX, USA). In detail, the DCFH-DA working solution was prepared using 10 μM serum-free medium and added to BV-2 cells for incubation for 30 min at 37 °C. Then, the cells were re-suspended in serum-free medium, and the fluorescence was read at the excitation and emission wavelengths of 500, 525 nm, respectively.

Calcein-AM/propidium iodide (PI) staining

BV-2 cells with different stimulations were collected and centrifuged at 1,000 g for 5 min at room temperature to collect cell pellet, which were rinsed in PBS. Next, BV-2 cells were dyed with 2 μM Calcein-AM and 4.5 μM PI provided by the assay kit (C2015M; Beyotime) at 37 °C for 30 min. A confocal laser scanning microscope (LSM800; Zeiss, Oberkosen, Germany) was applied to take the relevant imgaes (Qiu et al., 2019).

Immunofluorescence

BV-2 cells with different treatment were washed with pre-warmed PBS and fixed with 4% paraformaldehyde (P0099; Beyotime, Beijing, China) for 10 min and then permeabilized with 0.2% Triton X-100 (ST1722; Beyotime, Beijing, China) for another 10 min at room temperature. For the brain tissues, the mice were sacrificed and perfused with saline and 4% paraformaldehyde in 100 mM PBS. The brain tissue was collected, fixed in 4% paraformaldehyde overnight and immersed in 30% sucrose for 48 h. Then the coronal sections were resected into a thickness of 20 μm and permeabilized in 0.1% Triton X-100 for 30 min. Confocal microscopy (LSM800; Zeiss, 25X lens) was performed to observe and record the signals of Iba-1, CD86, and CD206.

Following washing with PBS, the brain tissues and BV-2 cells were incubated with the blocking buffer at 4 °C for 1 h and the primary antibodies against Iba-1 (019-19741; 1:1,000, Wako Fujifilm, Osaka, Japan), CD86 (942-RBM-4-P1; 1:1,000, Thermo Fisher Scientific) and CD206 (CL594-60143; 1:100, Proteintech, Wuhan, China) at 4 °C overnight. Subsequently, Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (ab150077; 1:1,000, Abcam, Cambridge, UK) was further used to incubate with the sections. The nuclei were finally counterstained with DAPI (C1002; Beyotime) and then the sections were rinsed in PBS three times and observed under a confocal laser microscope. The affiliated software was applied to calculate the relative mean fluorescence intensity (MFI).

Quantification of the oxidative stress indicators

The blood samples were collected by opening the thoracic cavity of the mice and centrifuged at 2,000 rpm for 10 min to harvest the plasm; meanwhile, the culture media of BV-2 cells in each group were harvested and centrifuged to collect the supernatant. The content of SOD and malondialdehyde (MDA) was quantified according to the instructions of the assay kits.

Quantitative PCR (qPCR)

A commercially available RNA isolation kit (RE-03113; Foregene, Beijing, China) was applied to separate total RNA from the brain tissue and cell samples. ChamQ Universal SYBR qPCR Master Mix (Q711-02; Vazyme, Nanjing, China) and Step One Plus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) were employed in PCR procedure (Sindhuja, Amuthalakshmi & Nalini, 2022). Primers used in this study were designed by PrimerBank and NCBI and validated with BLAST algorithm. Relative gene expression was calculated with the 2^−ΔΔCt method (Livak & Schmittgen, 2001). The sequences used were listed in Table 1.

Western blot

Total protein from BV-2 cells of each group was lysed and extracted using the lysis buffer containing phosphate and protease inhibitor (P0013C; Beyotime) and the concentration was quantified using BCA protein assay kit (BL521A; Biosharp, Hefei, China). Subsequently, equivalent volume of protein sample was separated in 12% SDS-PAGE separation gel and transferred to PVDF membrane, which was blocked by 5% bovine serum albumin (BSA). Next, the film was incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at room temperature for 1 h. After rinsing in TBST, the Tanon 5200 Multi Gel Imaging Analysis System (Tanon, Shanghai, China) was applied for the detection, and the results were processed with Gel-Pro Analyzer software 4.0 (Media Cybernetics, Rockville, Maryland, MD, USA). Relevant antibodies used were listed in Table 2.

Statistical analysis

Data from at least three independent trials were expressed as mean ± standard deviation. GraphPad Prism 6.0 was applied for statistical analyses. We tested the data for normality to ensure that the data used met the assumption of a normal distribution. Notably, by using Student’s t-test in order to be used for comparison of differences between two groups, while one-way ANOVA was used for comparison of differences between three and more groups. A P-value lower than 0.05 were regarded as statistically significant.

Cirsilineol restored the cognition function of the POCD mice

An anesthesia/surgery-induced mice model was established to investigate the potential effect of cirsilineol on POCD. The trajectories of the mice in each group during the Morris water maze test were displayed in Fig. 1A, and the escape latency, swimming speed and times of crossing island were also recorded. Following the modeling procedure, a longer escape latency on day 1 to day 4 and fewer times of island crossing were observed (Figs. 1B, 1D, P < 0.01) but the swimming speed was not affected (Fig. 1C, P > 0.05). However, the intervention of cirsilineol reduced the escape latency and increased the times of crossing island (Figs. 1B, 1D, P < 0.01) but did not evidently affect the swimming speed (Fig. 1C, P > 0.05). After the modeling procedure, the results of Y-maze test showed that the starting arm (SA) rate of the mice in each group was evidently reduced (Fig. 1E, P < 0.0001), while the administration of cirsilineol promoted the SA rate in the mice (Fig. 1E, P < 0.001). Collectively, these results indicated the potential of cirsilineol on the restoration of the cognition functions in the POCD model mice.

Cirsilineol repressed the microglial activation and oxidative stress in the POCD mice

To determine the activation and polarization state of microglia in POCD and to explore the role of cirsilineol in its regulation, the immunofluorescence assay was used to evaluate the activation of microglia using the Iba-1 antibody and to quantify the expression levels of relevant microglia markers (Cd86 for M1 and Cd206 for M2). As shown by an intense red fluorescence, the modeling resulted in a sharp elevation of Iba-1⁺ cells, (Figs. 2A, 2B, P < 0.0001), which was consistent with the upregulated expression of Cd86 (Fig. 2C, P < 0.01). The expression level of Cd206 was almost unchanged (Fig. 2D, P > 0.05). The intervention using cirsilineol reduced Iba-1⁺ cells (Figs. 2A, 2B, P < 0.0001), downregulated Cd86 expression but upregulated Cd206 expression in the POCD mice (Figs. 2C, 2D, P < 0.01).

The extent of oxidative stress in vivo was tested based on the quantified results on the levels of anti-oxidant indicator SOD and the peroxidation product MDA. It was found that the POCD modeling lowered the level of SOD but increased that of MDA (Figs. 2E, 2F, P < 0.01), whereas cirsilineol suppressed the generation of MDA but increased that of SOD in the POCD mice (Figs. 2E, 2F, P < 0.05). These results suggested the potentials of using cirsilineol to inhibit microglial activation and oxidative stress in POCD.

Cirsilineol promoted the survival of H₂O₂-treated microglia but suppressed ROS generation

Then H₂O₂-treated microglia were used to further examine the anti-inflammatory effects of Cirsilineol. The results confirmed the inhibitory effects of H₂O₂ on the viability of BV-2 cells (Fig. 3A, P < 0.0001) and its promoting effects on the production of ROS in BV-2 cells (Fig. 3B, P < 0.0001). However, cirsilineol, restored the viability of BV-2 cells and suppressed ROS production (Figs. 3A, 3B, P < 0.0001). Considering the efficiency of restoring the cell viability, 20 μM cirsilineol was applied in subsequent assays. The results from Calcein-AM/PI staining revealed a pro-apoptosis effect of H₂O₂ in BV-2 cells, as shown by increased fluorescence (Fig. 3C), which, however, was reduced by the treatment of cirsilineol (Fig. 3C). Collectively, these results indicated the pro-survival and anti-ROS potential of cirsilineol in H₂O₂-treated microglia.

Cirsilineol suppressed the microglial M1 polarization and oxidative stress in H₂O₂-treated microglia

Next, we based an in vitro cellular model to explore the effects of cirsilinel on microglia polarization and oxidative stress. Similar to the in vivo results, the exposure of microglia BV-2 to H₂O₂ has led to an increase in the MFI of CD86 (Figs. 4A, 4C, P < 0.0001), while that of CD206 was not affected (Figs. 4B, 4D, P > 0.05). A reduced MFI of CD86 but an enhanced MFI of CD206 in H₂O₂-treated microglia were observed following the intervention of cirsilineol (Figs. 4A–4D, P < 0.001). Also, H₂O₂ exposure evidently reduced the content of SOD and significantly increased the level of MDA in the microglia (Figs. 4E, 4F, P < 0.01), while such a trend was reversed by the treatment of cirsilineol (Figs. 4E, 4F, P < 0.01).

Cirsilineol modulated JAK/STAT pathway in H₂O₂-treated microglia

To explore the potential neuroprotective effects of cirsilinelo on POCD, we probed the mechanism of its regulation of the JAK/STAT signaling pathway. Here, we calculated the phosphorylation degrees of relevant proteins (JAK1, STAT1, and STAT6) of JAK/STAT pathway in H₂O₂-treated microglia. The phosphorylation levels of JAK1 and STAT1 were increased following H₂O₂ exposure (Figs. 5A, 5B, P < 0.01), whereas the level STAT6 was not affected significantly (Figs. 5A, 5B, P > 0.05). After the treatment of cirsilineol, the phosphorylation levels of JAK1 and STAT1 were reduced but that of STAT6 was increased evidently (Figs. 5A, 5B, P < 0.01). It could be concluded that cirsilineol modulated JAK/STAT pathway in H₂O₂-exposed microglia.